Circulating cell-free DNA from blood plasma of cancer patients can be used to non-invasively interrogate somatic tumor alterations. Here we develop MSK-ACCESS (Memorial Sloan Kettering - Analysis of Circulating cfDNA to Examine Somatic Status), an NGS assay for detection of very low frequency somatic alterations in 129 genes. Analytical validation demonstrated 92% sensitivity in de-novo mutation calling down to 0.5% allele frequency and 99% for a priori mutation profiling. To evaluate the performance of MSK-ACCESS, we report results from 681 prospective blood samples that underwent clinical analysis to guide patient management. Somatic alterations are detected in 73% of the samples, 56% of which have clinically actionable alterations. The utilization of matched normal sequencing allows retention of somatic alterations while removing over 10,000 germline and clonal hematopoiesis variants. Our experience illustrates the importance of analyzing matched normal samples when interpreting cfDNA results and highlights the importance of cfDNA as a genomic profiling source for cancer patients.
Cell free DNA (cfDNA) and circulating tumor cell free DNA (ctDNA) from blood (plasma) are increasingly being used in oncology for diagnosis, monitoring response, identifying cancer causing mutations and detecting recurrences. Circulating tumor RB1 DNA (ctDNA) is found in the blood (plasma) of retinoblastoma patients at diagnosis before instituting treatment (naïve). We investigated ctDNA in naïve unilateral patients before enucleation and during enucleation (6 patients/ 8 mutations with specimens collected 5–40 minutes from severing the optic nerve) In our cohort, following transection the optic nerve, ctDNA RB1 VAF was measurably lower than pre-enucleation levels within five minutes, 50% less within 15 minutes and 90% less by 40 minutes.
Circulating cell-free DNA (cfDNA) from blood plasma of cancer patients can be used to interrogate somatic tumor alterations non-invasively or when adequate tissue is unavailable. We have developed and clinically implemented MSK-ACCESS (Analysis of Circulating cfDNA to Evaluate Somatic Status), an NGS assay for detection of very low frequency somatic alterations in select exons and introns of 129 genes. Analytical validation demonstrated 92% sensitivity in de-novo mutation calling down to 0.5% allele frequency and 99% for a priori mutation profiling. To evaluate the performance and utility of MSK-ACCESS, we report results from the first 681 prospective blood samples (617 patients) that underwent clinical analysis to guide patient management. Somatic mutations, copy number, and/or structural variants were detected in 73% of the samples, and 56% of these circulating-tumor DNA (ctDNA) positive samples had clinically actionable alterations. The utilization of matched white blood cell sequencing allowed retention of somatic alterations while filtering out over 10,000 germline and clonal hematopoiesis variants, thereby greatly enhancing the specificity of the assay. Taken together, our experience illustrates the importance of analyzing a matched normal sample when interpreting cfDNA results and highlights the potential of cfDNA profiling to guide treatment selection, monitor treatment response, and identify mechanisms of treatment resistance.
Introduction: Circulating cell-free DNA (cfDNA) is a source of tumor-derived DNA to interrogate somatic alterations when tissue is not available or of insufficient quantity for analysis. At MSKCC, we have developed and validated MSK-ACCESS (Analysis of Circulating cfDNA to Evaluate Somatic Status), a targeted next-generation sequencing assay that can detect ultra-low frequency somatic variants in select exons and introns of 129 genes. MSK-ACCESS can identify mutations, copy number alterations, gene fusions, and MSI status in plasma and was recently approved by the NYS-DOH for clinical testing. Here, we present the results of the validation study and our clinical experience with MSK-ACCESS since June 2019. Methods: Target regions from 129 genes were selected to maximize coverage of actionable, oncogenic, and hotspot mutations based on the first 25,000 tumors sequenced using MSK-IMPACT, our institutional clinical sequencing assay. Plasma cfDNA and buffy-coat DNA were extracted from whole blood collected in cell-stabilizing tubes (STRECK BCT cell-free DNA tube). Unique molecular indexes were introduced during DNA library construction, allowing for error suppression from consensus reads collapsed by Marianas, an in-house-developed algorithm. These consensus reads enable variant calling at low allelic frequency (AF) based on a 10−6 background error rate. Results: Analytical validation of MSK-ACCESS demonstrated 93% accuracy (n=100 variants), 99% precision (n=153 variants), and 100% sensitivity based on an assay limit of detection of 0.5% AF (n=19 variants). Variants were detected down to 0.1% AF. To date, 240 clinical cfDNA and matched normal DNA pairs have been sequenced, analyzed for somatic alterations, and clinically reported to guide patient management. Most clinical cases were from lung (55%) or prostate (13%) cancers and submitted for diagnostic purposes (71%). Median raw coverage was 18,367X, and median consensus coverage was 1411X. Mutations were detected in 180 (75%) samples with a median variant AF of 1.8% (0.02% - 95%). Comparison of concurrent commercial plasma testing results to MSK-ACCESS revealed multiple variants that were of clonal hematopoiesis or germline origin incorrectly reported as somatic variants. In the lung cohort, 48 patients had tissue testing with MSK-IMPACT; among 32 patients with a driver alteration detected by MSK-ACCESS, 91% had the identical driver alteration reported by MSK-IMPACT. Additionally, MSK-ACCESS identified a MET exon 14 alteration in one lung cancer patient that led to protocol enrollment and partial response. Conclusions: Liquid biopsy testing using MSK-ACCESS reliably detected clinically actionable mutations, reducing the need for multiple biopsies. These results also illustrate the importance of analyzing a matched normal sample when interpreting cfDNA results and highlight the potential of using cfDNA analysis to guide treatment selection, assess for treatment response, and identify mechanisms of treatment resistance. This abstract is also being presented as Poster A20. Citation Format: A. Rose Brannon, Gowtham Jayakumaran, Monica Diosdado, Yu Hu, Anna Razumova, Fanli Meng, Emily Lebow, Juber Patel, Ian Johnson, Preethi Srinivasan, Maysun Hasan, Jenna-marie Dix, Aijazuddin Syed, Brian Houck-Loomis, Bob T. Li, Charles Rudin, David Solit, Marc Ladanyi, Maria Arcila, Dana Tsui, Ahmet Zehir, Michael Berger, Ryma Benayed. Validation and clinical implementation of MSK-ACCESS, an ultra-deep sequencing assay for noninvasive somatic mutation profiling [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr PR08.
A noted clinical application for liquid biopsy is as a non-invasive method of detecting and monitoring of minimal residual disease (MRD) in patients with cancer. The low concentration of circulating tumor DNA in blood, especially in early stage cancers, however, complicates the detection of tumor-derived cell-free DNA. To address this challenge, we have developed MSK-ACCESS (Analysis of Circulating cfDNA to Examine Somatic Status), a custom NGS assay covering selected exons from 129 cancer related genes for high-sensitivity detection of somatic mutations from plasma. Using ultra-high depth sequencing, with duplex unique molecular indexing (UMI), unique dual sample barcodes, and background error suppression, MSK-ACCESS is able to detect low-frequency (0.1%) variants with high confidence. The design of MSK-ACCESS leverages our dataset of more than 30,000 tumors profiled by our institutional tumor sequencing assay, MSK-IMPACT, ensuring that the majority of patients harbor multiple mutations that can be tracked in plasma. We have validated MSK-ACCESS using plasma samples collected from 40 healthy individuals and 70 cancer patients harboring a range of somatic mutations in 11 genes. Greater than 95% of mutations at allele fractions >0.1% were empirically detected, and we established the performance characteristics of the assay through intra- and inter-assay reproducibility tests and dilution experiments. We have initiated clinical trials in multiple tumor types to evaluate the benefit of early therapeutic intervention in patients where MSK-ACCESS can detect circulating tumor DNA following surgery. Tumor mutations revealed by MSK-IMPACT in surgically resected specimens will be monitored at regular intervals as evidence of MRD. As a proof of concept, we have applied MSK-ACCESS to monitor variants known from tissue tumor sequencing in pre- and post-surgical cfDNA samples from 9 colon adenocarcinoma patients. All samples were sequenced to an average total depth of approximately 20,000X coverage and subsequently collapsed to consensus sequences exhibiting an average noise level less than 0.0006%. Circulating tumor DNA was detected in 66%(6/9) of the pre-surgical samples. Of these samples, ctDNA was also detected in 50% (3/6) of the post-surgical samples. Overall, this study shows that MSK-ACCESS can be used to successfully detect MRD. Citation Format: Maysun M. Hasan, Juber Patel, Ian Johnson, Fanli Meng, Grittney K. Tam, Xiaohong Jing, Julie L. Yang, A. Rose Brannon, Jayakumaran Gowtham, Dennis P. Stephens, Monica Diosdado, Ryma Benayed, Ahmet Zehir, Chin-Tung Chen, Martin R. Weiser, Dana Tsui, Brian Houck-Loomis, Michael Berger. Tracking minimal residual disease in post-operative cell-free DNA using MSK-ACCESS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1387.
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